A simple and reliable method to prepare semen for transmission electron microscopy

Human semen is routinely analyzed when evaluating male infertility, particularly regarding parameters of sperm morphology and motility. Ordinary sample preparation usually follows several centrifugation steps, so any subsequent analysis performed by transmission electron microscopy (TEM) should avoid further damage of the samples. Herein we report a simple and reproducible method to prepare sperm for TEM that preserves the sample and avoid several centrifugations, speeding up the whole process. Forty seminal specimens were analysed (N= 20, healthy patients; N= 20, patients submitted to varicocelectomy). Half part of the samples was included in 1% agarose gel block and half part was processed without gel block inclusion. The infiltration of sperm in the gel block allows to achieve an optimal concentration of the sample in a narrow and well defined area. Gel blocks can thus be easily handled during dehydration, avoiding centrifugation. Resin embedment of the sample maintains it in a well-defined position, favouring ultrathin sectioning. The present method preserves sample structural integrity and morphology and improves final data interpretation.